Analysis of Juglans nigra Phloem Fungal Communities

Aaron Onufrak

Introduction

Background

Thousand cankers disease (TCD) is caused by the fungal pathogen Geosmithia morbida and its insect vector Pityophthorous juglandis (walnut twig beetle; WTB). The most susceptible TCD host is Juglans nigra (eastern black walnut) an important timber and nut crop native to portions of the eastern and midwestern U.S. TCD has been detected in 15 U.S. states and portions of Italy.

At this time, the effects of G. morbida infection on the J. nigra microbiome are not well understood. Therefore, the objective of this study was to examine the effects of G.moribida infection on the J. nigra phloem microbial communities. We expected that the fungal communities associated with J. nigra phloem tissues would shift in response to G. morbida infection.

Study Design

In the nursery compound at the University of Tennessee Knoxville trees were treated with the following three treatments with 24 trees per treatment:

  1. Water Control
  2. Phospho-Jet (Phosphorous Acid-based Fungicide, Arborjet)
  3. Rootshield (Trichoderma afroharzianum strain T-22, BioWorks)

Of the 72 trees, 36 trees were inoculated with the fungal pathogen Geosmithia morbida isolate TN17 at four separate, equally spaced locations along the stem. This isolate was chosen because it was originally isolated from black walnut in TN and its genome has been sequenced. The remaining 36 trees were inoculated with a potato dextrose agar (PDA) plug to use as an inoculation control. Inoculations took place 7 days following application of Phospho-Jet, Rootshield, and water treatments. On days 14, 28, and 56 post treatment application, trees were destructively sampled with roots and stem tissues sampled for DNA and RNA extraction.

Sample Processing

DNA was extracted from phloem tissues using a CTAB method. Amplified for the ITS2 region using the primers described in Cregger et al. 2018 and sequenced on the Illumina MiSeq platform using 2x250 bp chemistry at the University of Tennessee Genomics Core.

Sequence Processing Methodology

Step 1: Load Packages

Loading in the packages required to complete the sequence processing and statistical analyses.

library(dada2)
## Loading required package: Rcpp
library(Biostrings)
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## Attaching package: 'BiocGenerics'
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##     get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
##     match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
##     Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
##     table, tapply, union, unique, unsplit, which.max, which.min
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## Attaching package: 'S4Vectors'
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library(ShortRead)
## Loading required package: BiocParallel
## Loading required package: Rsamtools
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library(reticulate)

Step 2: Path Designations

Designating the path for the directory that contains the raw ITS2 sequence data and creating two objects, one for forward paths and the other for reverse paths.

# Creating an object that has the pathway for the sequence directory
phloem_its_path<-"/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing"

# Listing the files present in the sequence directory
list.files(phloem_its_path)
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## [300] "phloem-tree-9-ITS_S11_L001_I1_001.fastq.gz"  
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## [303] "phloem-tree-9-ITS_S11_L001_R2_001.fastq.gz"
# Creating objects for the forward and reverse reads
forwardreads_its_phloem<-sort(list.files(phloem_its_path, pattern="L001_R1_001.fastq",full.names=TRUE))

reversereads_its_phloem<-sort(list.files(phloem_its_path, pattern="L001_R2_001.fastq",full.names=TRUE))

# Checking number of forward and reverse files. Should be an equal number of forward and reverse files. 
length(forwardreads_its_phloem)
## [1] 75
length(reversereads_its_phloem)
## [1] 75
# Generating initial forward and reverse read quality plots
initial_forward_qual<-plotQualityProfile(forwardreads_its_phloem, aggregate=TRUE)
initial_forward_qual

initial_reverse_qual<-plotQualityProfile(forwardreads_its_phloem, aggregate=TRUE)
initial_reverse_qual

Step 3: Filtering of Ambiguous Reads

Removing reads with ambiguous bases.

# Creating an object  that strips away the pathway information leaving behind only the filename.
filebasename_its_phloem<-basename(forwardreads_its_phloem)

# Cleaning sample names to match with metadata sheet
basename_its_phloem<-sub("-ITS.*","",filebasename_its_phloem)
treebasename_its_phloem<-sub("phloem-","",basename_its_phloem)
numericbasename_its_phloem<-gsub("-","_",treebasename_its_phloem)

# Creating an object that contains only sample names.
samplenames_its_phloem<-numericbasename_its_phloem

# Creating two path objects that will store the paths for filtered forward and reverse reads. 
forward.filtN_its_phloem<-file.path(phloem_its_path,"filtN",basename(forwardreads_its_phloem))
reverse.filtN_its_phloem<-file.path(phloem_its_path,"filtN",basename(reversereads_its_phloem))

# Filtering forward and reverse reads using the filterAndTrim function. Specifying maxN=0 will indicate that sequences with at least 1 ambiguous base will be removed from the dataset. 
filterAndTrim(forwardreads_its_phloem,forward.filtN_its_phloem,reversereads_its_phloem,reverse.filtN_its_phloem,maxN=0)

# Creating quality profiles for the pre-filtered forward and reverse reads. 
filtn_qualplot_fwd_its_phloem<-plotQualityProfile(forward.filtN_its_phloem, aggregate=TRUE)
filtn_qualplot_fwd_its_phloem

filtn_qualplot_rev_its_phloem<-plotQualityProfile(reverse.filtN_its_phloem, aggregate=TRUE)
filtn_qualplot_rev_its_phloem

Step 4: Primer Removal

Removing primers used in the first step of PCR to amplify the ITS2 region. These are low diversity section that will not help with distinguishing between taxa.

# Creating strings that contain the forward and reverse primers used in the study.
its3ngs1<-"CATCGATGAAGAACGCAG"
its3ngs2<-"CAACGATGAAGAACGCAG"
its3ngs3<-"CACCGATGAAGAACGCAG"
its3ngs4<-"CATCGATGAAGAACGTAG"
its3ngs5<-"CATCGATGAAGAACGTGG"
its3ngs10<-"CATCGATGAAGAACGCTG"
its4ngr<-"TCCTSCGCTTATTGATATGC"
archits4<-"TCCTCGCCTTATTGATATGC"

# Creating a function called allorientations to identify all potential orientations of the primers. Then use the complement, reverse, and reverseComplement functions to store all possible orientations of the primer in the orientations object). Last, use the sapply function to convert all orientations into individual strings of text. 

allorientations<-function(primer){       
  require(Biostrings)     
  dna<-DNAString(primer)                                                    
  orientations<-c(Forward=dna, Complement=Biostrings::complement(dna),Reverse=Biostrings::reverse(dna), 
             RevComp=Biostrings::reverseComplement(dna)) 
  return(sapply(orientations,toString))
}

# Storing all possible orientations of primers in objects for each primer.
its3ngs1.ori<-allorientations(its3ngs1)
its3ngs2.ori<-allorientations(its3ngs2)
its3ngs3.ori<-allorientations(its3ngs3)
its3ngs4.ori<-allorientations(its3ngs4)
its3ngs5.ori<-allorientations(its3ngs5)
its3ngs10.ori<-allorientations(its3ngs10)
its4ngr.ori<-allorientations(its4ngr)
archits4.ori<-allorientations(archits4)

# Identifying occurrences of the primers by creating a function called primeroccurences.This function will use the vcountPattern function which will return a vector containing the number of times a particular primer is detected in a sequence and the readFastq function which will take all fastq files in a particular director and turn them into a single object.  
primeroccurences<-function(primer, directory) { 
  nhits<-vcountPattern(primer, sread(readFastq(directory)),fixed=FALSE) 
  return(sum(nhits>0))
}

# Using the sapply function to apply the primeroccurencesfunction to the directories that contain the forward and reverse reads.The rbind function creates a table for each combination of primer and forward and reverse reads. 
primertable_its_phloem<-
  rbind(its3ngs1.forwardreads_its_phloem=sapply(its3ngs1.ori,primeroccurences,directory=forward.filtN_its_phloem),
      its3ngs1.reversereads_its_phloem=sapply(its3ngs1.ori,primeroccurences,directory=reverse.filtN_its_phloem),
      its3ngs2.forwardreads_its_phloem=sapply(its3ngs2.ori,primeroccurences,directory=forward.filtN_its_phloem),
      its3ngs2.reversereads_its_phloem=sapply(its3ngs2.ori,primeroccurences,directory=reverse.filtN_its_phloem),
      its3ngs3.forwardreads_its_phloem=sapply(its3ngs3.ori,primeroccurences,directory=forward.filtN_its_phloem),
      its3ngs3.reversereads_its_phloem=sapply(its3ngs3.ori,primeroccurences,directory=reverse.filtN_its_phloem),
      its3ngs4.forwardreads_its_phloem=sapply(its3ngs4.ori,primeroccurences,directory=forward.filtN_its_phloem),
      its3ngs4.reversereads_its_phloem=sapply(its3ngs4.ori,primeroccurences,directory=reverse.filtN_its_phloem),
      its3ngs5.forwardreads_its_phloem=sapply(its3ngs5.ori,primeroccurences,directory=forward.filtN_its_phloem),
      its3ngs5.reversereads_its_phloem=sapply(its3ngs5.ori,primeroccurences,directory=reverse.filtN_its_phloem),
      its3ngs10.forwardreads_its_phloem=sapply(its3ngs10.ori,primeroccurences,directory=forward.filtN_its_phloem),
      its3ngs10.reversereads_its_phloem=sapply(its3ngs10.ori,primeroccurences,directory=reverse.filtN_its_phloem),
      its4ngr.forwardreads_its_phloem=sapply(its4ngr.ori,primeroccurences,directory=forward.filtN_its_phloem),
      its4ngr.reversereads_its_phloem=sapply(its4ngr.ori,primeroccurences,directory=reverse.filtN_its_phloem),
      archits4.forwardreads_its_phloem=sapply(archits4.ori,primeroccurences,directory=forward.filtN_its_phloem),
      archits4.reversereads_its_phloem=sapply(archits4.ori,primeroccurences,directory=reverse.filtN_its_phloem))
primertable_its_phloem
##                                   Forward Complement Reverse RevComp
## its3ngs1.forwardreads_its_phloem  4197059          0       0       2
## its3ngs1.reversereads_its_phloem      241          0       0     335
## its3ngs2.forwardreads_its_phloem  1629310          0       0       0
## its3ngs2.reversereads_its_phloem       72          0       0     166
## its3ngs3.forwardreads_its_phloem  1247251          0       0       2
## its3ngs3.reversereads_its_phloem      107          0       0     140
## its3ngs4.forwardreads_its_phloem   542977          0       0       0
## its3ngs4.reversereads_its_phloem       39          0       0     532
## its3ngs5.forwardreads_its_phloem    10281          0       0       0
## its3ngs5.reversereads_its_phloem        1          0       0      60
## its3ngs10.forwardreads_its_phloem   89814          0       0       0
## its3ngs10.reversereads_its_phloem       9          0       0      31
## its4ngr.forwardreads_its_phloem       264          0       0     789
## its4ngr.reversereads_its_phloem   3631996          0       0       0
## archits4.forwardreads_its_phloem      356          0       0     895
## archits4.reversereads_its_phloem  4130887          0       0       0
# Creating a directory to store the forward and reverse reads after they have been trimmed. 
path.cut<-file.path(phloem_its_path,"cutadapt")
if(!dir.exists(path.cut)) dir.create(path.cut)

forwardreads.cut_its_phloem<-file.path(path.cut,basename(forwardreads_its_phloem))
reversereads.cut_its_phloem<-file.path(path.cut,basename(reversereads_its_phloem))

# Creating objects containing the forward and reverse primer reverse compliment strings.The rc function takes a sequence object provided by the user and creates the reverse compliment of the sequence. 

its3ngs1.rc1<-dada2::rc(its3ngs1)
its3ngs2.rc1<-dada2::rc(its3ngs2)
its3ngs3.rc1<-dada2::rc(its3ngs3)
its3ngs4.rc1<-dada2::rc(its3ngs4)
its3ngs5.rc1<-dada2::rc(its3ngs5)
its3ngs10.rc1<-dada2::rc(its3ngs10)
its4ngr.rc1<-dada2::rc(its4ngr)
archits4.rc1<-dada2::rc(archits4)

# Using the paste function we then create objects that contain the flags for the potential combinations of each forward and reverse primer and the reverse compliment. These flags will serve as the argumentst that we provide to cutadapt. The ^ at the beginning of the sequence indicates that the primer should be removed from the beginning of the sequence. 

its3ngs1.its4.r1.flags<-paste("-a"," ", "^", its3ngs1,"...",its4ngr.rc1, sep='') 
its3ngs1.arch.r1.flags<-paste("-a"," ", "^", its3ngs1,"...",archits4.rc1, sep='') 
its3ngs2.its4.r1.flags<-paste("-a"," ", "^", its3ngs2,"...",its4ngr.rc1, sep='') 
its3ngs2.arch.r1.flags<-paste("-a"," ", "^", its3ngs2,"...",archits4.rc1, sep='') 
its3ngs3.its4.r1.flags<-paste("-a"," ", "^", its3ngs3,"...",its4ngr.rc1, sep='') 
its3ngs3.arch.r1.flags<-paste("-a"," ", "^", its3ngs3,"...",archits4.rc1, sep='')
its3ngs4.its4.r1.flags<-paste("-a"," ", "^", its3ngs4,"...",its4ngr.rc1, sep='') 
its3ngs4.arch.r1.flags<-paste("-a"," ", "^", its3ngs4,"...",archits4.rc1, sep='')
its3ngs5.its4.r1.flags<-paste("-a"," ", "^", its3ngs5,"...",its4ngr.rc1, sep='') 
its3ngs5.arch.r1.flags<-paste("-a"," ", "^", its3ngs5,"...",archits4.rc1, sep='')
its3ngs10.its4.r1.flags<-paste("-a"," ", "^", its3ngs10,"...",its4ngr.rc1, sep='') 
its3ngs10.arch.r1.flags<-paste("-a"," ", "^", its3ngs10,"...",archits4.rc1, sep='')

its4.its3ngs1.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs1.rc1, sep='') 
arch.its3ngs1.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs1.rc1, sep='') 
its4.its3ngs2.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs2.rc1, sep='') 
arch.its3ngs2.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs2.rc1, sep='') 
its4.its3ngs3.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs3.rc1, sep='') 
arch.its3ngs3.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs3.rc1, sep='') 
its4.its3ngs4.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs4.rc1, sep='') 
arch.its3ngs4.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs4.rc1, sep='') 
its4.its3ngs5.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs5.rc1, sep='') 
arch.its3ngs5.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs5.rc1, sep='') 
its4.its3ngs10.r2.flags<-paste("-A"," ", "^", its4ngr,"...",its3ngs10.rc1, sep='') 
arch.its3ngs10.r2.flags<-paste("-A"," ", "^", archits4,"...",its3ngs10.rc1, sep='') 

# Specifying the conda environment to use for cutadapt
use_condaenv(condaenv="/Users/aarononfurak/anaconda3/envs/cutadaptenv/bin/python")

# Using cutadapt to remove the primers from each read.
for (i in seq_along(forwardreads_its_phloem)){
  system2("conda",args=c("run -n cutadaptenv cutadapt",
                          its3ngs1.its4.r1.flags,
                          its3ngs1.arch.r1.flags,
                          its3ngs2.its4.r1.flags,
                          its3ngs2.arch.r1.flags,
                          its3ngs3.its4.r1.flags,
                          its3ngs3.arch.r1.flags,
                          its3ngs4.its4.r1.flags,
                          its3ngs4.arch.r1.flags,
                          its3ngs5.its4.r1.flags,
                          its3ngs5.arch.r1.flags,
                          its3ngs10.its4.r1.flags,
                          its3ngs10.arch.r1.flags,
                          its4.its3ngs1.r2.flags,
                          arch.its3ngs1.r2.flags,
                          its4.its3ngs2.r2.flags,
                          arch.its3ngs2.r2.flags,
                          its4.its3ngs3.r2.flags,
                          arch.its3ngs3.r2.flags,
                          its4.its3ngs4.r2.flags,
                          arch.its3ngs4.r2.flags,
                          its4.its3ngs5.r2.flags,
                          arch.its3ngs5.r2.flags,
                          its4.its3ngs10.r2.flags,
                          arch.its3ngs10.r2.flags,
                          "--discard-untrimmed","--minimum-length",10,"--report=minimal",
                          "-o",forwardreads.cut_its_phloem[i], "-p",reversereads.cut_its_phloem[i],
                          forward.filtN_its_phloem[i],reverse.filtN_its_phloem[i]))
}

# Evaluting the efficacy of primer removal with cutadapt using the primeroccurrences function.
primertable_its_phloem_2<-
  rbind(its3ngs1.forwaredreads.cut_its_phloem=sapply(its3ngs1.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
      its3ngs1.reversereads.cut_its_phloem=sapply(its3ngs1.ori,primeroccurences,directory=reversereads.cut_its_phloem),
      its3ngs2.forwaredreads.cut_its_phloem=sapply(its3ngs2.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
      its3ngs2.reversereads.cut_its_phloem=sapply(its3ngs2.ori,primeroccurences,directory=reversereads.cut_its_phloem),
      its3ngs3.forwaredreads.cut_its_phloem=sapply(its3ngs3.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
      its3ngs3.reversereads.cut_its_phloem=sapply(its3ngs3.ori,primeroccurences,directory=reversereads.cut_its_phloem),
      its3ngs4.forwaredreads.cut_its_phloem=sapply(its3ngs4.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
      its3ngs4.reversereads.cut_its_phloem=sapply(its3ngs4.ori,primeroccurences,directory=reversereads.cut_its_phloem),
      its3ngs5.forwaredreads.cut_its_phloem=sapply(its3ngs5.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
      its3ngs5.reversereads.cut_its_phloem=sapply(its3ngs5.ori,primeroccurences,directory=reversereads.cut_its_phloem),
      its3ngs10.forwaredreads.cut_its_phloem=sapply(its3ngs10.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
      its3ngs10.reversereads.cut_its_phloem=sapply(its3ngs10.ori,primeroccurences,directory=reversereads.cut_its_phloem),
      its4ngr.forwaredreads.cut_its_phloem=sapply(its4ngr.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
      its4ngr.reversereads.cut_its_phloem=sapply(its4ngr.ori,primeroccurences,directory=reversereads.cut_its_phloem),
      archits4.forwaredreads.cut_its_phloem=sapply(archits4.ori,primeroccurences,directory=forwardreads.cut_its_phloem),
      archits4.reversereads.cut_its_phloem=sapply(archits4.ori,primeroccurences,directory=reversereads.cut_its_phloem))
primertable_its_phloem_2
##                                        Forward Complement Reverse RevComp
## its3ngs1.forwaredreads.cut_its_phloem      102          0       0       0
## its3ngs1.reversereads.cut_its_phloem         0          0       0       0
## its3ngs2.forwaredreads.cut_its_phloem       11          0       0       0
## its3ngs2.reversereads.cut_its_phloem         0          0       0       0
## its3ngs3.forwaredreads.cut_its_phloem       56          0       0       0
## its3ngs3.reversereads.cut_its_phloem         0          0       0       0
## its3ngs4.forwaredreads.cut_its_phloem       11          0       0       0
## its3ngs4.reversereads.cut_its_phloem         0          0       0       0
## its3ngs5.forwaredreads.cut_its_phloem        0          0       0       0
## its3ngs5.reversereads.cut_its_phloem         0          0       0       0
## its3ngs10.forwaredreads.cut_its_phloem       3          0       0       0
## its3ngs10.reversereads.cut_its_phloem        0          0       0       0
## its4ngr.forwaredreads.cut_its_phloem         0          0       0       0
## its4ngr.reversereads.cut_its_phloem         32          0       0       0
## archits4.forwaredreads.cut_its_phloem        0          0       0       0
## archits4.reversereads.cut_its_phloem        17          0       0       0
# Comparing the pre vs post primer detections.
primertable_its_phloem
##                                   Forward Complement Reverse RevComp
## its3ngs1.forwardreads_its_phloem  4197059          0       0       2
## its3ngs1.reversereads_its_phloem      241          0       0     335
## its3ngs2.forwardreads_its_phloem  1629310          0       0       0
## its3ngs2.reversereads_its_phloem       72          0       0     166
## its3ngs3.forwardreads_its_phloem  1247251          0       0       2
## its3ngs3.reversereads_its_phloem      107          0       0     140
## its3ngs4.forwardreads_its_phloem   542977          0       0       0
## its3ngs4.reversereads_its_phloem       39          0       0     532
## its3ngs5.forwardreads_its_phloem    10281          0       0       0
## its3ngs5.reversereads_its_phloem        1          0       0      60
## its3ngs10.forwardreads_its_phloem   89814          0       0       0
## its3ngs10.reversereads_its_phloem       9          0       0      31
## its4ngr.forwardreads_its_phloem       264          0       0     789
## its4ngr.reversereads_its_phloem   3631996          0       0       0
## archits4.forwardreads_its_phloem      356          0       0     895
## archits4.reversereads_its_phloem  4130887          0       0       0
primertable_its_phloem_2
##                                        Forward Complement Reverse RevComp
## its3ngs1.forwaredreads.cut_its_phloem      102          0       0       0
## its3ngs1.reversereads.cut_its_phloem         0          0       0       0
## its3ngs2.forwaredreads.cut_its_phloem       11          0       0       0
## its3ngs2.reversereads.cut_its_phloem         0          0       0       0
## its3ngs3.forwaredreads.cut_its_phloem       56          0       0       0
## its3ngs3.reversereads.cut_its_phloem         0          0       0       0
## its3ngs4.forwaredreads.cut_its_phloem       11          0       0       0
## its3ngs4.reversereads.cut_its_phloem         0          0       0       0
## its3ngs5.forwaredreads.cut_its_phloem        0          0       0       0
## its3ngs5.reversereads.cut_its_phloem         0          0       0       0
## its3ngs10.forwaredreads.cut_its_phloem       3          0       0       0
## its3ngs10.reversereads.cut_its_phloem        0          0       0       0
## its4ngr.forwaredreads.cut_its_phloem         0          0       0       0
## its4ngr.reversereads.cut_its_phloem         32          0       0       0
## archits4.forwaredreads.cut_its_phloem        0          0       0       0
## archits4.reversereads.cut_its_phloem        17          0       0       0

Step 5: Quality Filtering

Quality filtering data prior to denoising with dada2. Generating quality plots for the sequences that have had their primers removed and use these quality plots to determine what parameters should be used to filter sequences.

# Creating new pathways for the quality filtered forward and reverse reads. 
filtforward_its_phloem<-file.path(phloem_its_path, "filtered", paste0(samplenames_its_phloem,"_F_filt.fastq.gz"))
filtreverse_its_phloem<-file.path(phloem_its_path,"filtered",paste0(samplenames_its_phloem,"_R_filt.fastq.gz"))

# Removing paths to non-existent negative control samples that lost all reads following initial quality filtering. 
forwardreads.cut_its_phloem_for_qual<-forwardreads.cut_its_phloem[grep("NTC|negative",forwardreads.cut_its_phloem,invert = TRUE)]
reversereads.cut_its_phloem_for_qual<-reversereads.cut_its_phloem[grep("NTC|negative",reversereads.cut_its_phloem,invert = TRUE)]

# Creating quality plot profiles for the primer trimmed forward and reverse reads. 
primercut_qualplot_fwd_its_phloem<-plotQualityProfile(forwardreads.cut_its_phloem_for_qual, aggregate=TRUE)
primercut_qualplot_fwd_its_phloem

primercut_qualplot_rev_its_phloem<-plotQualityProfile(reversereads.cut_its_phloem_for_qual, aggregate=TRUE)
primercut_qualplot_rev_its_phloem

# Quality filteirng reads using a maximum expected error threshold of 2 for the forward and reverse reads. Based on the quality plots setting trunlen to 200. This will truncate all reads to 200 bp. The output will be two directories containing filtered forward and filtered reverse reads. 
filter.out_its_phloem<-filterAndTrim(fwd=forwardreads.cut_its_phloem, filt=filtforward_its_phloem, rev=reversereads.cut_its_phloem, filt.rev=filtreverse_its_phloem, maxN=0, maxEE=c(2,2), compress=TRUE,truncLen = 200, multithread = FALSE)
## The filter removed all reads: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/1st_AMP_NTC_F_filt.fastq.gz and /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/1st_AMP_NTC_R_filt.fastq.gz not written.
## Warning in file.remove(fout[[1]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/1st_AMP_NTC_F_filt.fastq.gz',
## reason 'No such file or directory'
## Warning in file.remove(fout[[2]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/1st_AMP_NTC_R_filt.fastq.gz',
## reason 'No such file or directory'
## The filter removed all reads: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/2nd_AMP_NTC2_F_filt.fastq.gz and /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/2nd_AMP_NTC2_R_filt.fastq.gz not written.
## Warning in file.remove(fout[[1]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/2nd_AMP_NTC2_F_filt.fastq.gz',
## reason 'No such file or directory'
## Warning in file.remove(fout[[2]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/2nd_AMP_NTC2_R_filt.fastq.gz',
## reason 'No such file or directory'
## The filter removed all reads: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/negative_F_filt.fastq.gz and /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/negative_R_filt.fastq.gz not written.
## Warning in file.remove(fout[[1]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/negative_F_filt.fastq.gz',
## reason 'No such file or directory'
## Warning in file.remove(fout[[2]]): cannot remove file
## '/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/negative_R_filt.fastq.gz',
## reason 'No such file or directory'
## Some input samples had no reads pass the filter.
# The previous step created an object called filter.out_its_phloem. This object is currently a matrix. Converting this object into a dataframe. 
filter.out_its_phloem<-as.data.frame(filter.out_its_phloem)

# Determining which sample lost the most number of reads during the filtering process. First creatin a new column that represents the differences between the reads that were fed into the filter and the reads that passed the filter. 
filter.out_its_phloem$diffs <- filter.out_its_phloem$reads.in-filter.out_its_phloem$reads.out

# Using the order command to sort the dataframe by the new column (diffs) that was created above. 
filter.out_its_phloem[order(filter.out_its_phloem$reads.out),]
##                                              reads.in reads.out diffs
## 1st-AMP-NTC-ITS_S20_L001_R1_001.fastq.gz            0         0     0
## 2nd-AMP-NTC2-ITS_S30_L001_R1_001.fastq.gz           0         0     0
## phloem-negative-ITS_S10_L001_R1_001.fastq.gz        0         0     0
## phloem-tree-11-ITS_S43_L001_R1_001.fastq.gz     27848      9811 18037
## phloem-tree-32-ITS_S24_L001_R1_001.fastq.gz     68178     38330 29848
## phloem-tree-22-ITS_S38_L001_R1_001.fastq.gz     89065     45526 43539
## phloem-tree-5-ITS_S18_L001_R1_001.fastq.gz      79999     49758 30241
## phloem-tree-29-ITS_S70_L001_R1_001.fastq.gz     65046     50941 14105
## phloem-tree-12-ITS_S74_L001_R1_001.fastq.gz     85740     53214 32526
## phloem-tree-4-ITS_S61_L001_R1_001.fastq.gz      78762     53425 25337
## phloem-tree-39-ITS_S58_L001_R1_001.fastq.gz    108120     54538 53582
## phloem-tree-64-ITS_S65_L001_R1_001.fastq.gz     91345     56178 35167
## phloem-tree-1-ITS_S52_L001_R1_001.fastq.gz      87412     58875 28537
## phloem-tree-16-ITS_S68_L001_R1_001.fastq.gz    104051     59413 44638
## phloem-tree-20-ITS_S60_L001_R1_001.fastq.gz    119264     59857 59407
## phloem-tree-2-ITS_S71_L001_R1_001.fastq.gz     125002     60354 64648
## phloem-tree-40-ITS_S32_L001_R1_001.fastq.gz    100625     62315 38310
## phloem-tree-41-ITS_S67_L001_R1_001.fastq.gz    113835     62497 51338
## phloem-tree-35-ITS_S3_L001_R1_001.fastq.gz     131301     62667 68634
## phloem-tree-23-ITS_S49_L001_R1_001.fastq.gz    103122     62733 40389
## phloem-tree-52-ITS_S47_L001_R1_001.fastq.gz     82271     62971 19300
## phloem-tree-31-ITS_S34_L001_R1_001.fastq.gz     92392     63036 29356
## phloem-tree-65-ITS_S69_L001_R1_001.fastq.gz    139614     64781 74833
## phloem-tree-25-ITS_S16_L001_R1_001.fastq.gz     85430     64842 20588
## phloem-tree-50-ITS_S42_L001_R1_001.fastq.gz    131925     65005 66920
## phloem-tree-9-ITS_S11_L001_R1_001.fastq.gz     106159     65019 41140
## phloem-tree-47-ITS_S63_L001_R1_001.fastq.gz     96692     65303 31389
## phloem-tree-61-ITS_S8_L001_R1_001.fastq.gz     100366     66863 33503
## phloem-tree-13-ITS_S2_L001_R1_001.fastq.gz     121481     68451 53030
## phloem-tree-26-ITS_S33_L001_R1_001.fastq.gz    122196     70153 52043
## phloem-tree-48-ITS_S9_L001_R1_001.fastq.gz     106880     70582 36298
## phloem-tree-34-ITS_S36_L001_R1_001.fastq.gz    105150     70922 34228
## phloem-tree-27-ITS_S12_L001_R1_001.fastq.gz    106197     71190 35007
## phloem-tree-18-ITS_S59_L001_R1_001.fastq.gz    119322     71224 48098
## phloem-tree-38-ITS_S37_L001_R1_001.fastq.gz    106100     71593 34507
## phloem-tree-43-ITS_S44_L001_R1_001.fastq.gz    118041     72383 45658
## phloem-tree-54-ITS_S41_L001_R1_001.fastq.gz    110060     72765 37295
## phloem-tree-6-ITS_S19_L001_R1_001.fastq.gz     101923     75019 26904
## phloem-tree-63-ITS_S7_L001_R1_001.fastq.gz     125931     75248 50683
## phloem-tree-7-ITS_S13_L001_R1_001.fastq.gz     125731     75263 50468
## phloem-tree-58-ITS_S25_L001_R1_001.fastq.gz    122170     75790 46380
## phloem-tree-49-ITS_S39_L001_R1_001.fastq.gz    118821     76268 42553
## phloem-tree-46-ITS_S15_L001_R1_001.fastq.gz    109375     76804 32571
## phloem-tree-55-ITS_S57_L001_R1_001.fastq.gz    106983     77083 29900
## phloem-tree-51-ITS_S51_L001_R1_001.fastq.gz    130904     77093 53811
## phloem-tree-70-ITS_S64_L001_R1_001.fastq.gz    113802     77651 36151
## phloem-tree-10-ITS_S56_L001_R1_001.fastq.gz    103002     77878 25124
## phloem-tree-72-ITS_S35_L001_R1_001.fastq.gz    104917     78975 25942
## phloem-tree-42-ITS_S28_L001_R1_001.fastq.gz    122718     79425 43293
## phloem-tree-53-ITS_S22_L001_R1_001.fastq.gz    111978     80129 31849
## phloem-tree-62-ITS_S62_L001_R1_001.fastq.gz    122146     81218 40928
## phloem-tree-57-ITS_S46_L001_R1_001.fastq.gz     98944     81828 17116
## phloem-tree-56-ITS_S66_L001_R1_001.fastq.gz    115947     81980 33967
## phloem-tree-17-ITS_S6_L001_R1_001.fastq.gz     127892     82143 45749
## phloem-tree-21-ITS_S14_L001_R1_001.fastq.gz    114310     82229 32081
## phloem-tree-19-ITS_S73_L001_R1_001.fastq.gz    108635     84202 24433
## phloem-tree-37-ITS_S1_L001_R1_001.fastq.gz     127970     84613 43357
## phloem-tree-28-ITS_S75_L001_R1_001.fastq.gz    119173     84900 34273
## phloem-tree-67-ITS_S45_L001_R1_001.fastq.gz    111190     85673 25517
## phloem-tree-68-ITS_S53_L001_R1_001.fastq.gz    117124     85837 31287
## phloem-tree-14-ITS_S54_L001_R1_001.fastq.gz    129158     86261 42897
## phloem-tree-3-ITS_S23_L001_R1_001.fastq.gz     132941     87889 45052
## phloem-tree-66-ITS_S40_L001_R1_001.fastq.gz    145075     87932 57143
## phloem-tree-69-ITS_S55_L001_R1_001.fastq.gz    123536     90120 33416
## phloem-tree-33-ITS_S50_L001_R1_001.fastq.gz    137983     90353 47630
## phloem-tree-60-ITS_S27_L001_R1_001.fastq.gz    134196     91095 43101
## phloem-tree-24-ITS_S29_L001_R1_001.fastq.gz    127910     93707 34203
## phloem-tree-15-ITS_S72_L001_R1_001.fastq.gz    128910     94590 34320
## phloem-tree-44-ITS_S48_L001_R1_001.fastq.gz    113987     95908 18079
## phloem-tree-45-ITS_S26_L001_R1_001.fastq.gz    122561     98891 23670
## phloem-tree-8-ITS_S17_L001_R1_001.fastq.gz     140443    102315 38128
## phloem-tree-36-ITS_S21_L001_R1_001.fastq.gz    168039    106297 61742
## phloem-tree-30-ITS_S5_L001_R1_001.fastq.gz     143247    110405 32842
## phloem-tree-59-ITS_S31_L001_R1_001.fastq.gz    153485    113092 40393
## phloem-tree-71-ITS_S4_L001_R1_001.fastq.gz     180930    133821 47109
# Removing paths to non-existent negative control samples that lost all reads following initial quality filtering. 
filtforward_its_phloem_no_missing<-filtforward_its_phloem[grep("NTC|negative",filtforward_its_phloem,invert = TRUE)]
filtreverse_its_phloem_no_missing<-filtreverse_its_phloem[grep("NTC|negative",filtreverse_its_phloem,invert = TRUE)]

# We then generate quality plots for the forward and reverse reads after quality filtering
filtered_qualplot_fwd_its_phloem<-plotQualityProfile(filtforward_its_phloem_no_missing, aggregate=TRUE)
filtered_qualplot_fwd_its_phloem

filtered_qualplot_rev_its_phloem<-plotQualityProfile(filtreverse_its_phloem_no_missing, aggregate=TRUE)
filtered_qualplot_rev_its_phloem

Step 6: Learn Error Rates

Learning the error rates of the data

# Learning errors for forward and reverse reads. 
errorforward_its_phloem<-learnErrors(filtforward_its_phloem_no_missing, multithread = TRUE)
## 101698600 total bases in 508493 reads from 8 samples will be used for learning the error rates.
errorreverse_its_phloem<-learnErrors(filtreverse_its_phloem_no_missing,multithread = TRUE)
## 101698600 total bases in 508493 reads from 8 samples will be used for learning the error rates.
# Error plots for forward & reverse reads
errplots_fwd_its_phloem<-plotErrors(errorforward_its_phloem, nominalQ=TRUE)
errplots_fwd_its_phloem
## Warning: Transformation introduced infinite values in continuous y-axis
## Transformation introduced infinite values in continuous y-axis

errplots_rev_its_phloem<-plotErrors(errorreverse_its_phloem,nominalQ = TRUE)
errplots_rev_its_phloem
## Warning: Transformation introduced infinite values in continuous y-axis
## Transformation introduced infinite values in continuous y-axis

Step 7: Dereplication

Dereplicating reads

# dereplication(merging identical reads into single sequences)
derepforward_its_phloem<-derepFastq(filtforward_its_phloem_no_missing,verbose=TRUE)
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_1_F_filt.fastq.gz
## Encountered 15359 unique sequences from 58875 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_10_F_filt.fastq.gz
## Encountered 11205 unique sequences from 77878 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_11_F_filt.fastq.gz
## Encountered 1355 unique sequences from 9811 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_12_F_filt.fastq.gz
## Encountered 8308 unique sequences from 53214 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_13_F_filt.fastq.gz
## Encountered 7877 unique sequences from 68451 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_14_F_filt.fastq.gz
## Encountered 23277 unique sequences from 86261 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_15_F_filt.fastq.gz
## Encountered 11470 unique sequences from 94590 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_16_F_filt.fastq.gz
## Encountered 9404 unique sequences from 59413 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_17_F_filt.fastq.gz
## Encountered 12082 unique sequences from 82143 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_18_F_filt.fastq.gz
## Encountered 9764 unique sequences from 71224 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_19_F_filt.fastq.gz
## Encountered 10266 unique sequences from 84202 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_2_F_filt.fastq.gz
## Encountered 10407 unique sequences from 60354 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_20_F_filt.fastq.gz
## Encountered 6154 unique sequences from 59857 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_21_F_filt.fastq.gz
## Encountered 13268 unique sequences from 82229 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_22_F_filt.fastq.gz
## Encountered 10086 unique sequences from 45526 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_23_F_filt.fastq.gz
## Encountered 15118 unique sequences from 62733 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_24_F_filt.fastq.gz
## Encountered 9766 unique sequences from 93707 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_25_F_filt.fastq.gz
## Encountered 6347 unique sequences from 64842 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_26_F_filt.fastq.gz
## Encountered 5351 unique sequences from 70153 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_27_F_filt.fastq.gz
## Encountered 8574 unique sequences from 71190 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_28_F_filt.fastq.gz
## Encountered 10134 unique sequences from 84900 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_29_F_filt.fastq.gz
## Encountered 7795 unique sequences from 50941 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_3_F_filt.fastq.gz
## Encountered 6862 unique sequences from 87889 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_30_F_filt.fastq.gz
## Encountered 12327 unique sequences from 110405 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_31_F_filt.fastq.gz
## Encountered 10381 unique sequences from 63036 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_32_F_filt.fastq.gz
## Encountered 7193 unique sequences from 38330 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_33_F_filt.fastq.gz
## Encountered 15310 unique sequences from 90353 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_34_F_filt.fastq.gz
## Encountered 11393 unique sequences from 70922 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_35_F_filt.fastq.gz
## Encountered 6508 unique sequences from 62667 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_36_F_filt.fastq.gz
## Encountered 12617 unique sequences from 106297 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_37_F_filt.fastq.gz
## Encountered 11129 unique sequences from 84613 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_38_F_filt.fastq.gz
## Encountered 11816 unique sequences from 71593 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_39_F_filt.fastq.gz
## Encountered 10191 unique sequences from 54538 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_4_F_filt.fastq.gz
## Encountered 4645 unique sequences from 53425 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_40_F_filt.fastq.gz
## Encountered 8298 unique sequences from 62315 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_41_F_filt.fastq.gz
## Encountered 7260 unique sequences from 62497 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_42_F_filt.fastq.gz
## Encountered 7447 unique sequences from 79425 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_43_F_filt.fastq.gz
## Encountered 7630 unique sequences from 72383 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_44_F_filt.fastq.gz
## Encountered 9419 unique sequences from 95908 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_45_F_filt.fastq.gz
## Encountered 9494 unique sequences from 98891 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_46_F_filt.fastq.gz
## Encountered 17548 unique sequences from 76804 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_47_F_filt.fastq.gz
## Encountered 11489 unique sequences from 65303 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_48_F_filt.fastq.gz
## Encountered 15333 unique sequences from 70582 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_49_F_filt.fastq.gz
## Encountered 11950 unique sequences from 76268 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_5_F_filt.fastq.gz
## Encountered 7909 unique sequences from 49758 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_50_F_filt.fastq.gz
## Encountered 5520 unique sequences from 65005 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_51_F_filt.fastq.gz
## Encountered 4779 unique sequences from 77093 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_52_F_filt.fastq.gz
## Encountered 6598 unique sequences from 62971 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_53_F_filt.fastq.gz
## Encountered 8556 unique sequences from 80129 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_54_F_filt.fastq.gz
## Encountered 15258 unique sequences from 72765 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_55_F_filt.fastq.gz
## Encountered 13325 unique sequences from 77083 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_56_F_filt.fastq.gz
## Encountered 12912 unique sequences from 81980 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_57_F_filt.fastq.gz
## Encountered 10589 unique sequences from 81828 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_58_F_filt.fastq.gz
## Encountered 11434 unique sequences from 75790 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_59_F_filt.fastq.gz
## Encountered 11556 unique sequences from 113092 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_6_F_filt.fastq.gz
## Encountered 12537 unique sequences from 75019 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_60_F_filt.fastq.gz
## Encountered 14445 unique sequences from 91095 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_61_F_filt.fastq.gz
## Encountered 13114 unique sequences from 66863 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_62_F_filt.fastq.gz
## Encountered 18230 unique sequences from 81218 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_63_F_filt.fastq.gz
## Encountered 10116 unique sequences from 75248 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_64_F_filt.fastq.gz
## Encountered 8030 unique sequences from 56178 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_65_F_filt.fastq.gz
## Encountered 9396 unique sequences from 64781 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_66_F_filt.fastq.gz
## Encountered 12037 unique sequences from 87932 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_67_F_filt.fastq.gz
## Encountered 8463 unique sequences from 85673 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_68_F_filt.fastq.gz
## Encountered 12596 unique sequences from 85837 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_69_F_filt.fastq.gz
## Encountered 15204 unique sequences from 90120 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_7_F_filt.fastq.gz
## Encountered 5449 unique sequences from 75263 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_70_F_filt.fastq.gz
## Encountered 19564 unique sequences from 77651 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_71_F_filt.fastq.gz
## Encountered 19252 unique sequences from 133821 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_72_F_filt.fastq.gz
## Encountered 8143 unique sequences from 78975 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_8_F_filt.fastq.gz
## Encountered 16555 unique sequences from 102315 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_9_F_filt.fastq.gz
## Encountered 9881 unique sequences from 65019 total sequences read.
derepreverse_its_phloem<-derepFastq(filtreverse_its_phloem_no_missing,verbose=TRUE)
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_1_R_filt.fastq.gz
## Encountered 11476 unique sequences from 58875 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_10_R_filt.fastq.gz
## Encountered 15459 unique sequences from 77878 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_11_R_filt.fastq.gz
## Encountered 3802 unique sequences from 9811 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_12_R_filt.fastq.gz
## Encountered 12324 unique sequences from 53214 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_13_R_filt.fastq.gz
## Encountered 14817 unique sequences from 68451 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_14_R_filt.fastq.gz
## Encountered 19376 unique sequences from 86261 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_15_R_filt.fastq.gz
## Encountered 18109 unique sequences from 94590 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_16_R_filt.fastq.gz
## Encountered 15731 unique sequences from 59413 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_17_R_filt.fastq.gz
## Encountered 14218 unique sequences from 82143 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_18_R_filt.fastq.gz
## Encountered 18101 unique sequences from 71224 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_19_R_filt.fastq.gz
## Encountered 16875 unique sequences from 84202 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_2_R_filt.fastq.gz
## Encountered 13953 unique sequences from 60354 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_20_R_filt.fastq.gz
## Encountered 18082 unique sequences from 59857 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_21_R_filt.fastq.gz
## Encountered 16072 unique sequences from 82229 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_22_R_filt.fastq.gz
## Encountered 14863 unique sequences from 45526 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_23_R_filt.fastq.gz
## Encountered 11629 unique sequences from 62733 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_24_R_filt.fastq.gz
## Encountered 24409 unique sequences from 93707 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_25_R_filt.fastq.gz
## Encountered 11709 unique sequences from 64842 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_26_R_filt.fastq.gz
## Encountered 16643 unique sequences from 70153 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_27_R_filt.fastq.gz
## Encountered 19714 unique sequences from 71190 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_28_R_filt.fastq.gz
## Encountered 19088 unique sequences from 84900 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_29_R_filt.fastq.gz
## Encountered 10894 unique sequences from 50941 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_3_R_filt.fastq.gz
## Encountered 23131 unique sequences from 87889 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_30_R_filt.fastq.gz
## Encountered 19593 unique sequences from 110405 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_31_R_filt.fastq.gz
## Encountered 11626 unique sequences from 63036 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_32_R_filt.fastq.gz
## Encountered 13465 unique sequences from 38330 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_33_R_filt.fastq.gz
## Encountered 20640 unique sequences from 90353 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_34_R_filt.fastq.gz
## Encountered 15450 unique sequences from 70922 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_35_R_filt.fastq.gz
## Encountered 18851 unique sequences from 62667 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_36_R_filt.fastq.gz
## Encountered 21949 unique sequences from 106297 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_37_R_filt.fastq.gz
## Encountered 18151 unique sequences from 84613 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_38_R_filt.fastq.gz
## Encountered 12350 unique sequences from 71593 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_39_R_filt.fastq.gz
## Encountered 19299 unique sequences from 54538 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_4_R_filt.fastq.gz
## Encountered 15685 unique sequences from 53425 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_40_R_filt.fastq.gz
## Encountered 13763 unique sequences from 62315 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_41_R_filt.fastq.gz
## Encountered 13242 unique sequences from 62497 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_42_R_filt.fastq.gz
## Encountered 23292 unique sequences from 79425 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_43_R_filt.fastq.gz
## Encountered 14923 unique sequences from 72383 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_44_R_filt.fastq.gz
## Encountered 13996 unique sequences from 95908 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_45_R_filt.fastq.gz
## Encountered 14597 unique sequences from 98891 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_46_R_filt.fastq.gz
## Encountered 14533 unique sequences from 76804 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_47_R_filt.fastq.gz
## Encountered 17717 unique sequences from 65303 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_48_R_filt.fastq.gz
## Encountered 12050 unique sequences from 70582 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_49_R_filt.fastq.gz
## Encountered 15890 unique sequences from 76268 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_5_R_filt.fastq.gz
## Encountered 9646 unique sequences from 49758 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_50_R_filt.fastq.gz
## Encountered 16975 unique sequences from 65005 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_51_R_filt.fastq.gz
## Encountered 21977 unique sequences from 77093 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_52_R_filt.fastq.gz
## Encountered 14714 unique sequences from 62971 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_53_R_filt.fastq.gz
## Encountered 21477 unique sequences from 80129 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_54_R_filt.fastq.gz
## Encountered 18268 unique sequences from 72765 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_55_R_filt.fastq.gz
## Encountered 17809 unique sequences from 77083 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_56_R_filt.fastq.gz
## Encountered 16392 unique sequences from 81980 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_57_R_filt.fastq.gz
## Encountered 13930 unique sequences from 81828 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_58_R_filt.fastq.gz
## Encountered 16995 unique sequences from 75790 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_59_R_filt.fastq.gz
## Encountered 23966 unique sequences from 113092 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_6_R_filt.fastq.gz
## Encountered 16922 unique sequences from 75019 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_60_R_filt.fastq.gz
## Encountered 16099 unique sequences from 91095 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_61_R_filt.fastq.gz
## Encountered 12702 unique sequences from 66863 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_62_R_filt.fastq.gz
## Encountered 16768 unique sequences from 81218 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_63_R_filt.fastq.gz
## Encountered 19514 unique sequences from 75248 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_64_R_filt.fastq.gz
## Encountered 16669 unique sequences from 56178 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_65_R_filt.fastq.gz
## Encountered 21161 unique sequences from 64781 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_66_R_filt.fastq.gz
## Encountered 19500 unique sequences from 87932 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_67_R_filt.fastq.gz
## Encountered 18048 unique sequences from 85673 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_68_R_filt.fastq.gz
## Encountered 17471 unique sequences from 85837 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_69_R_filt.fastq.gz
## Encountered 17574 unique sequences from 90120 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_7_R_filt.fastq.gz
## Encountered 23454 unique sequences from 75263 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_70_R_filt.fastq.gz
## Encountered 14791 unique sequences from 77651 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_71_R_filt.fastq.gz
## Encountered 27822 unique sequences from 133821 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_72_R_filt.fastq.gz
## Encountered 20311 unique sequences from 78975 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_8_R_filt.fastq.gz
## Encountered 14875 unique sequences from 102315 total sequences read.
## Dereplicating sequence entries in Fastq file: /Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sequence_processing/filtered/tree_9_R_filt.fastq.gz
## Encountered 13642 unique sequences from 65019 total sequences read.
# Assigning the sample names to the dereplicated sequence objects
names(derepforward_its_phloem)<-samplenames_its_phloem[grep("AMP|negative",samplenames_its_phloem,invert = TRUE)]
names(derepreverse_its_phloem)<-samplenames_its_phloem[grep("AMP|negative",samplenames_its_phloem,invert = TRUE)]

Step 8: Denoising with DADA2

Now that reads have been quality filtered, error rates have been learned, and reads are dereplicated, proceeding to the sequence inference step of the dada2 pipeline.

#denoise with dada2
dadaforwardreads_its_phloem<-dada(derepforward_its_phloem,err=errorforward_its_phloem,multithread = TRUE,pool=TRUE)
dadareversereads_its_phloem<-dada(derepreverse_its_phloem,err=errorreverse_its_phloem,multithread = TRUE,pool=TRUE)

Step 9: Merge Reads and Chimera Removal

Merging reads and removing chimeric sequences

# First forward and reverse reads are merged using mergePairs. 
merge_its_phloem<-mergePairs(dadaforwardreads_its_phloem,filtforward_its_phloem_no_missing,dadareversereads_its_phloem,filtreverse_its_phloem_no_missing,verbose=TRUE)
## 13143 paired-reads (in 404 unique pairings) successfully merged out of 58688 (in 968 pairings) input.
## 74909 paired-reads (in 802 unique pairings) successfully merged out of 77668 (in 1168 pairings) input.
## 9615 paired-reads (in 224 unique pairings) successfully merged out of 9801 (in 279 pairings) input.
## 16728 paired-reads (in 498 unique pairings) successfully merged out of 53061 (in 934 pairings) input.
## 18816 paired-reads (in 429 unique pairings) successfully merged out of 68326 (in 817 pairings) input.
## 40490 paired-reads (in 1039 unique pairings) successfully merged out of 85887 (in 1849 pairings) input.
## 87445 paired-reads (in 726 unique pairings) successfully merged out of 94451 (in 1131 pairings) input.
## 41053 paired-reads (in 403 unique pairings) successfully merged out of 59290 (in 688 pairings) input.
## 14978 paired-reads (in 268 unique pairings) successfully merged out of 82003 (in 626 pairings) input.
## 24391 paired-reads (in 511 unique pairings) successfully merged out of 71091 (in 1022 pairings) input.
## 79106 paired-reads (in 772 unique pairings) successfully merged out of 84001 (in 1123 pairings) input.
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# I then create a sequence table
seqtab_its_phloem<-makeSequenceTable(merge_its_phloem)
hist(nchar(colnames(seqtab_its_phloem)))

# I then remove chimeras using the removeBimeraDenovo function.
seqtab.nochim_its_phloem<-removeBimeraDenovo(seqtab_its_phloem,method="consensus", multithread=TRUE, verbose=TRUE)
## Identified 2480 bimeras out of 4432 input sequences.
#Number of ASVs in the study
ncol((seqtab.nochim_its_phloem))
nrow(seqtab.nochim_its_phloem)

#Number of sequences
sum(seqtab.nochim_its_phloem)

Step 10: Taxonomy Assignment

Assiging taxonomy to sequences from the UNITE database using a modified UNITE database fasta file that includes the ITS2 sequence of Juglans nigra accession MF182372.1.

# Assigning taxonomy using the assignTaxonomy function.
taxa_its_phloem<-assignTaxonomy(seqtab.nochim_its_phloem,"/Volumes/control_experiments/phosphojet_and_rootshield_experiments_summer_2020/nursery_summer_2020/phloem_fungi_ornl_jnigrarnaseq_su20_ajo_v2/sh_general_release_dynamic_25.07.2023_dev.fasta",multithread = TRUE, minBoot=80)
## UNITE fungal taxonomic reference detected.
# Converting the taxonomy assignments to data frame so we can filter the taxonomic assignments.
taxa.original_its_phloem<-as.data.frame(taxa_its_phloem)

# Selecting only ASVs assigned to a fungal kingdom
taxa.no.plant<-taxa.original_its_phloem[grep("Fungi",taxa.original_its_phloem$Kingdom,invert=FALSE),]

# Filtering out ASVs assigned to a fungal phyla
taxa.na.omit_its_phloem<-taxa.no.plant[-(which(is.na(taxa.no.plant$Phylum))),]

Step 11: Taxonomy Merging and ASV Filtering

The last step is to filter the ASV abundance table by merging the ASV table with the taxonomy assignments. This will remove ASVs from the count table that were not assigned to a fungal phyla or were identified as plant sequences.

# Transposing the ASV table so that taxonomy can be added. 
t.seqtab.nochim_its_phloem<-t(seqtab.nochim_its_phloem)

# Merging the two tables together based on row name. 
t.seqtab.nochim.filt_its_phloem<-t.seqtab.nochim_its_phloem[row.names(t.seqtab.nochim_its_phloem)%in%row.names(taxa.na.omit_its_phloem),]

# Number of ASVs & number of sequences post-filtering
nrow(t.seqtab.nochim.filt_its_phloem)
## [1] 1515
sum(t.seqtab.nochim.filt_its_phloem)
## [1] 2855172
# Merging taxonomy information into ASV table
t.seqtab.tax_its_phloem<-merge(t.seqtab.nochim.filt_its_phloem,taxa.na.omit_its_phloem, by="row.names")

sort(colSums(t.seqtab.nochim.filt_its_phloem))
##  tree_5 tree_41 tree_11 tree_38 tree_50 tree_49  tree_1 tree_60 tree_40  tree_9 
##    3894    7516    8979    9045    9321   10770   11421   12214   12265   12317 
## tree_31 tree_12 tree_17 tree_35 tree_48 tree_13 tree_58 tree_62 tree_34 tree_70 
##   12393   12709   13494   14124   16646   16773   18297   18404   19313   20658 
## tree_63 tree_18 tree_61 tree_23  tree_2 tree_22  tree_4 tree_66 tree_56 tree_64 
##   20964   21614   24159   25006   26653   26798   26945   28608   28646   29015 
## tree_36 tree_32 tree_14 tree_37 tree_20 tree_39 tree_27 tree_43 tree_21 tree_29 
##   29197   30968   31678   32796   33015   34009   35757   37503   37559   39372 
## tree_16 tree_72 tree_46 tree_52 tree_65 tree_54 tree_47 tree_55 tree_42  tree_6 
##   39435   41835   43306   43823   44070   45626   46441   51111   53650   54781 
## tree_25 tree_26 tree_53  tree_7  tree_3 tree_69 tree_10  tree_8 tree_28 tree_51 
##   56451   56603   58128   58468   59725   60107   60832   61745   63494   63832 
## tree_24 tree_68 tree_33 tree_67 tree_57 tree_19 tree_15 tree_30 tree_44 tree_59 
##   64976   65526   65890   69661   70371   70683   79794   83804   84519   87883 
## tree_45 tree_71 
##   91249   96538
# Creating ASV labels and make these new row names.
asvnumber_its_phloem<-as.character(c(1:nrow(t.seqtab.nochim.filt_its_phloem)))
asvnumber_its_phloem<-paste("asv_its",labels(asvnumber_its_phloem))

row.names(t.seqtab.tax_its_phloem)<-NULL
row.names(t.seqtab.tax_its_phloem)<-asvnumber_its_phloem

# Pulling out ASV's that are geosmithia
geosmithia.seqs.table<-t.seqtab.tax_its_phloem[grep("Geosmithia",t.seqtab.tax_its_phloem$Genus),]

# Pulling out ASV labels and sequences
geosmithia.seqs<-geosmithia.seqs.table[,1,drop=FALSE]

# Converting the table to a vector object where the ASV names are assigned to the appropriate sequence.
geosmithia.seqs.fmt<-setNames(geosmithia.seqs$Row.names,row.names(geosmithia.seqs))

# Converting to DNA string set for export as a fasta. 
geosmithia.seqs.string.set<-Biostrings::DNAStringSet(geosmithia.seqs.fmt)

Biostrings::writeXStringSet(geosmithia.seqs.string.set,"~/Google Drive/My Drive/utk_project/phospho_jet_rootshield_manuscript/manuscript_versions/manuscript_v01/analyses/r_sequence_processing_data/geosmithia.asvs.fa")

write.table(t.seqtab.tax_its_phloem,"~/Google Drive/My Drive/utk_project/phospho_jet_rootshield_manuscript/manuscript_versions/manuscript_v01/analyses/r_sequence_processing_data/feb_22_2024.t.seqtab.tax_its_phloem_manuscript_version.txt",col.names=TRUE,sep='\t',row.names=TRUE)

save.image("~/Google Drive/My Drive/utk_project/phospho_jet_rootshield_manuscript/manuscript_versions/manuscript_v01/analyses/r_sequence_processing_data/feb_22_2024_its_phloem_sequence_processing_manuscript_version.RData")